The present invention relates to a whey protein hydrolysate having specific physicochemical properties and a method for producing the same, and in particular to a novel whey protein hydrolysate which as exceptional gastrointestinal absorption properties and amino acid balance, which is effective in preventing and treating food allergies and has antioxidant action, which is palatable, and which can be used in a wide variety of applications, as well as to a method for producing the same.
The present invention also relates to a method in which a peptide mixture containing a specific amount of a free amino acid is produced with consistent quality in a state that is always stable.
The present invention furthermore relates to a method in which a peptide mixture with a low phenylalanine content that can be ingested daily by patients suffering from amino acid metabolic disorder, particularly, phenylketonuria, which is a disease requiring a limited intake of phenylalanine, is produced with consistent quality in a state that is always stable.
In the present specifications, percentages are based on weight, except for transmittance and inhibiting ratios, unless otherwise specified.
It has recently become clear that, in terms of digestion, oligopeptides have better absorption rates and a better balance of amino acids following absorption than do mixtures of free amino acids (Rakuno Kagaku-Shokuhin no Kenkyu [Dairy Sciences and Food Research], Vol. 39, No. A p. 283 (1990)). It is also clear that there has been a rapid increase in patients suffering from allergies induced by food proteins, and that many allergies caused by whey protein, particularly xcex2-lactoglobulin, have appeared, particularly in infants (Rakuno Kagaku-Shokuhin no Kenkyu [Daily Sciences and Food Research], Vol. 39, No. A, p. 283 (1990)). There is a need to reduce the antigenicity of whey protein in food products for infants or to essentially remove whey protein antigen from food products for infants.
The hydrolysis of whey protein has been widely adopted as a means for reducing the antigenicity of whey protein in food products for infants or for essentially removing whey protein antigen from food products for infants, but hydrolysates with an extremely low percentage of free amino acids often taste bad, which can cause problems when they are ingested. Hydrolysates of whey protein are also sometimes unstable against heat, resulting in precipitation in a liquid state, browning, and other such disadvantages which have posed problems when conventional hydrolysates are used as oral nutrients and the like.
Preventing oxidation is another major issue when whey protein hydrolysates are used in food products, particularly in fatty foods (such as powder milk prepared for infants, which contains as much as 27% fat per 100 g). That is, the balance between saturated and unsaturated fatty acids is considered from a nutritional standpoint in food products that contain fats, but a drawback is that unsaturated d fatty acids are readily oxidized. DHA and the like which are contained in large amounts in the biological membranes of brain, neural, and retinal tissue and which have recently been believed to play a role in the manifestation of their functions release an extremely strong oxidation odor once they are oxidized, having a markedly adverse effect on product quality, and there is thus a need to prevent their further oxidation.
Ingested amino acids are degraded by transglutaminase, glutamate dehydrogenase, and the like to produce ammonia, but the ammonia thus produced is toxic and must be immediately treated by the liver, so it is essential that no ingested foods contain ammonia.
In this regard, it is extremely important that whey protein hydrolysates contain no ammonia.
In view of the nitrogen equilibrium in mature animals, nitrogen should be ingested in an amount corresponding to the minimum metabolic amount of nitrogen, but nitrogen is ineffective when just given in the form of ammonia, and it must be ingested in the form of essential amino acids. Foods that are ingested must therefore contain the necessary amounts of essential amino acids.
Many methods for producing hydrolysates by the enzymolysis of whey proteins have been developed in view of the nutritional and physiochemical background of the proteins and amino acids described above. Several examples are given below.
1) Whey protein is hydrolyzed either with two enzymes, one being a Bacillus subtilis-derived endopeptidase and one being trypsin, or with three enzymes, including a Bacillus subtillis-derived endopeptidase, trypsin, and chymotrypsin, to obtain an oligopeptide mixture with a molecular weight of no more than 2,000 daltons, antigen persistence of no more than 10xe2x88x924, and a free acid content of no more than 5% (Japanese Laid-Open Patent Publication 4-248959).
2) Whey protein is hydrolyzed with an alkali protease to obtain a hydrolysate which has at least 75 mol % dipeptides and tripeptides, which has a free amino acid content of less than 5%, which consists of at least four amino acids, and which has less than 20 mol % peptides with an average chain length of 6.2 (Japanese Laid-Open Patent Publication 5-505304).
3) Whey, casein, and soybeans are hydrolyzed with pepsin, trypsin, and chymotrypsin, and ultrafiltration of the product results in an oligopeptide which has a molecular weight of no more than 60,000 daltons and which is 40 to 60% peptides containing 4 to 10 amino acids (Japanese Laid-Open Patent Publication 3-187348).
4) Whey protein is hydrolyzed by thermal denaturation at a pH of 6 to 10 and a temperature of 60 to 80xc2x0 C., and the enzyme is inactivated by heat to obtain a hydrolysate which has a molecular weight of no more than 10,000 daltons, main peaks of 1,000 to 5,000, an average peptide chain length of 3 to 8, a free amino acid content of no more than 20%, and no more than {fraction (1/10,000)} xcex2-lactoglobulin antigenicity (Japanese Laid-Open Patent Publication 4-112753).
5) Whey protein is hydrolyzed with trypsin, xcex1-chymotrypsin, and Aspergillus and Bacillus enzymes to obtain a low allergenic peptide with a molecular weight of no more than 10,000 daltons and the capacity to induce oral immunotolerance (Japanese Laid-Open Patent Publication 5-5000).
6) Casein is hydrolyzed with an acidic protease and is hydrolyzed with a neutral peptidase to obtain a peptide with a molecular weight of no more than 3,000 daltons, a free amino acid content of 30 to 55%, no more than 1 in 10,000 parts xcex1s-casein in an ELISA inhibition test for xcex1s-casein, and a 5% solution bitterness organoleptic value no greater than that corresponding to a 0.04% aqueous solution of caffeine (Japanese Laid-Open Patent Publication 6-113893).
7) A method has been disclosed in which whey is hydrolyzed at a pH of 5 to 11 using a neutral protease (Aspergillus) and then heated at a pH of 2 to 4, and the precipitate is removed to obtain 50% dipeptides and tripeptides (Japanese Patent Publication 5-82412).
Peptide mixtures obtained by the proteolysis of animal proteins (animal milk, eggs, meat, fish, and the like) or vegetable proteins (soybeans, wheat, and the like) are known to have properties such as thickening, foaming, antioxidant, digestive, mineral solubilizing, and low antigenicity properties, as well as epithelial cell growth factor, cell growth factor, calcium absorption promoting function, opioid-like activity, and other such physiologically active functions (Shokuhin to Kaihatsu [Food Products and Development], Vol. 26, No. 11, pp. 28-36 (1991)). They are an indispensable material in the manufacture of meat, fish paste, breads, sweets, mineral fortified food products, infant food products, sports beverages, general health foods, enteric nutrients, food products to combat protein allergies, special nutritional food products, medical drugs, and the like.
Methods for producing the peptide mixtures used in the manufacture of these food products and medical drugs vary, depending on the application, and can be broadly divided into:
a) methods for producing the target peptide mixture by hydrolyzing the starting material protein with only an endopeptidase to minimize the production of free amino acids;
b) methods for hydrolyzing the starting material protein with a combination of an endopeptidase and an exopeptidase to produce a peptide mixture which conversely contains a prescribed amount of free amino acids;
c) methods for purifying and fractionating such peptide mixtures into a target peptide mixture by separation such as ultrafiltration (UV), reverse osmosis (RO), gel filtration, and ion exchange resin methods; and the like.
Phenylketonuria (henceforth PKU) is a congenital metabolic disorder in which phenylalanine (henceforth Phe) accumulates in the blood, resulting in neurological disorders and developmental disorders, due to a congenital deficiency of phenylalanine hydroxylase which converts the amino acid phenylalanine into tyrosine. Patients suffering from PKU must accordingly strictly limit the amounts of Phe ingested under the supervision of a physician so as to avoid the accumulation of Phe in the body.
Since, on the other hand, Phe is a common amino acid that is usually contained in an amount of about 3 to 5% in proteins, patients of PKU have had to ingest part or all of the protein component of food products or infant milk preparations by substituting them with amino acid mixtures containing no Phe. These types of amino acid mixtures, however, suffer from drawbacks such as the disagreeable taste characteristic of amino acids and the diarrhea which results from high osmotic pressure in the intestines. There is thus a desire on the part of patients, their families, and physicians for a palatable source of protein serving as a suitable food therapy for patients of PKU.
A method involving the use of xcexa-casein glycomacropeptide (henceforth GMP) as a protein source for patients of PKU has been disclosed as one such method (Japanese Laid-Open Patent Publication 4-126051).
The amino acid sequence of GMP contains no Phe, the molecular weight is a substantial 8,000 daltons, and the problem of elevated osmotic pressure is virtually absent, making this substance an effective source of protein for patients of PKU. However, the isolation of GMP is extremely complicated and is unsuitable for industrial production. Moreover, recent nutritional findings have made it clear that oligopeptides are more readily digested than proteins.
Another example of the use of oligopeptides as a source of protein for patients of PKU is the method in which proteolysis is brought about with a protease, fractions containing no Phe are recovered by gel filtration, and the resulting low phenylalanine peptide (henceforth LPP) is used (Journal of Food Science, Vol. 41, pp. 1029-1032 (1976), and Japanese Laid-Open Patent Publication 61-68426).
In another method that has been disclosed, proteins are treated with an exopeptidase or are treated with an exopeptidase following treatment with an endopeptidase, activated carbon is used to adsorb polypeptides containing virtually no aromatic amino acids as well as low molecular weight peptides having terminal free aromatic amino acids or aromatic amino acids, and the low molecular weight substances are separated using reverse osmosis membranes or ion exchange electrodialysis membranes to produce LPP (Japanese Patent Publication 2-54070). This method has made the industrial manufacture of LPP possible.
The following conventional techniques are examples for producing peptides by the hydrolysis of starting material proteins using protease.
1) A method that has been disclosed is characterized in that enzymolysis is brought about for 0.5 to 10 hours in an aqueous system containing both an endo-type protease and exo-type protease to obtain oligopeptides with an average chain length of 3 to 10 and with very little bitterness (Japanese Laid-Open Patent Publication 62-143697).
2) In another method that has been disclosed, any starter protein starting material is dispersed to between 5 and 20% (w/v) in water, the pH is adjusted to between 1 and 4 with an acid, and enzymolysis is brought about as two or more acidic proteases are added simultaneously or consecutively to suppress the production of free amino acids for 8 to 72 hours at a temperature of 25 to 60xc2x0 C., so as to produce a low molecular weight peptide composition (Japanese Patent Publication 57-45560).
3) A method for producing a casein partial hydrolysate by controlling the flow ratio of the casein solution has been disclosed for methods involving the partial hydrolysis of a casein solution using a column packed with an immobilized enzyme (Japanese Patent Publication 3-31421).
4) A method for producing a proteolytic product in which the production of insoluble products is prevented is characterized in that a dissolving promoter and a protein or a substance containing a protein are mixed and dissolved in dissolving water or the like, and one or more proteases is or are added to bring about digestion and thus produce a proteolytic product, during which time the viscosity of the solution obtained following the addition of at least the initial proteolytic product is measured over time, and the digestion reaction is stopped before the viscosity begins to fall following a substantial increase (Japanese Patent Publication 3-58252).
5) A method for inactivating the enzyme by heat treatment when the ratio of hydrolysis reaches 5 to 25% has been disclosed for methods of producing peptide mixtures which are the protease hydrolysates of animal milk xcexa-casein-derived glycopolypeptide, with a Fisher value ranging from 30 to 60 (Japanese Laid-Open Patent Publication 2-300137).
6) A method for measuring peptide concentration with a peptide sensor and a method for measuring the average chain length of peptides have been disclosed for methods of producing hydrolysates in the hydrolysis of proteins using immobilized proteases (edited by Shokuhin Sangyo Baioriakuta Shisutemu Gijutsu Kenkyu Kumiai [Association for Research on Food Industry Bioreactor Systems], Jissen Baioriakuta [Practical Bioreactors], pp. 166-184, published by Association for Research on Food Industry Bioreactor Systems (1990)).
7) It has been disclosed that gluten hydrolysates with excellent foaming stability can be produced by measuring the hydrophobicity of gluten hydrolysates using reverse phase high performance liquid chromatography (high performance liquid chromatography is henceforth HPLC) in methods for producing gluten hydrolysates by hydrolyzing wheat gluten using immobilized proteases (Jissen Baioriakuta [Practical Bioreactors], pp. 106-126, published by Association for Research on Food Industry Bioreactor Systems (1990)).
8) It is known that the free amino acid content in the target product is measured during processing and management of the fermentation of amino acids such as lysine and glutamic acid and the production of fermented foods such as soy sauce and miso sauce (Shokuhin Kogyo [Food Industry], Vol. 34, No. 16, pp. 1-11 (1991)).
9) A method has been disclosed for producing a low molecular weight peptide with no antigenicity, a molecular weight of no more than 1,000 daltons, a free amino acid content of no more than 20%, and an aromatic amino acid content of no more than 1.0% of the total amino acids by recovering the peptide components through gel filtration following hydrolysis until the starting material protein is no longer found to be antigenic and until the aromatic amino acids contained in the starting material protein are at least 90% free amino acids (Japanese Laid-Open Patent Publication 2-138991).
10) A method that has been disclosed for producing a peptide mixture from cow milk whey protein is characterized in that cow milk whey protein is brought into contact with a protease capable of simulating the digestion of proteins occurring in the body, so as to continue the hydrolysis until the product contains virtually no residual protein, that is, until it contains no nitrogen capable of precipitating in 12% trichloroacetic acid, and until a peptide mixture is obtained in which at least 50% of the peptides contain 2 to 5 amino acids, with a free amino acid content of no more than 15% (Japanese Patent Publication 62-61039).
11) A cosmetic and topical skin agent that has been disclosed is characterized in that the hydrolysate of a protein has a molecular weight of no more than 1,000, with at least 90% of the aromatic amino acids being free amino acids, has action in activating the growth of human skin cells, and has no lactoprotein antigenicity (Japanese Laid-Open Patent Publication 4-26604).
Although decreases in the antigenicity of the whey protein hydrolysate, improvements in the taste, the free amino acid content, the molecular weight distribution, or the like are considered in the aforementioned conventional techniques, no consideration has been given to the ammonia content and the anitoxidant action of the whey protein hydrolysate. A drawback in the past has thus been that whey protein hydrolysates could not be used for a wide range of food products.
Moreover, as indicated in the aforementioned conventional techniques, when peptide mixtures are produced by hydrolyzing starting material proteins with proteases, the end point of the hydrolysis reaction has been determined by measuring the hydrolysate viscosity, ratio of hydrolysis, degree of hydrophobicity, or the like using the reaction time, the protein solution flow ratio, and the like as indices, but it is extremely difficult in these methods to achieve an accuratio grasp of the changing physicochemical state of the hydrolysate, particularly the free amino acid content, and a fatal drawback which needs to be remedied in conventional methods for producing peptide mixtures is that each batch which is manufactured results in a peptide mixture with a different free amino acid content and composition, leading to peptide mixtures of inconsistent quality.
Furthermore, to bring about enzyme reaction with good reproducibility, it is necessary to strictly control the reaction temperature, pH, enzyme titer, substratio concentration, and the like, which is impractical for operations on an industrial scale and makes it extremely difficult for practical purposes to obtain a consistent free amino acid ratio.
This means that the production of high quality LPP is also plagued by the following fatal drawbacks. As described above, in LPP manufacturing methods which include a step involving the hydrolysis of proteins using proteases, the method for lowering the Phe content of the peptide mixture is based on the principle that a sufficient amount of Phe is freed from the proteins by the hydrolysis of the proteins using the protease, and that the freed Phe is removed by gel filtration, activated carbon adsorption, or the like, which means the control of the protease-based hydrolysis of the protein is an important technique for producing high quality LPP.
That is, when Phe is insufficiently freed, a low amount of Phe is removed by gel filtration or activated carbon treatment following the enzymolysis, resulting in a peptide mixture with a higher Phe content, which is unsuitable for use by patients suffering from PKU. When too much Phe is freed (in other words, when enzymolysis has progressed too far), there is a higher ratio of free amino acids other than Phe, resulting in extremely poor taste, and other drawbacks include extremely poor therapeutic effects because of the unpleasant taste, the incidence of diarrhea, and the like, so these problems currently need to be resolved. That is, an extremely important issue for producing high quality LPP is to consistently ensure a certain content of free Phe in peptide mixtures obtained by the hydrolysis of proteins using proteases.
The inventors conducted painstaking research in view of the foregoing and perfected the present invention upon discovering a palatable whey protein hydrolysate that is obtained by the hydrolysis of whey protein, that is effective in avoiding, preventing, and treating food allergies, that is readily digested, that has a low ammonia content, that has antioxidant action, and that can be used in a wide range of applications, as well as a method for producing the same.
In view of the above, and as a result of painstaking research on a method for producing a peptide mixture of consistent quality in a state that is always stable in light of the aforementioned conventional techniques, the inventors perfected the present invention upon discovering that the desired peptide mixture is obtained by measuring briefly and over time the amount of a specific amino acid that is freed in the hydrolysate as a result of hydrolysis, by calculating the proportion with the amount of said specific amino acid contained in the starting material protein, and by immediately stopping the hydrolysis when the value falls within a specific range.
Furthermore, in view of the above, and as a result of painstaking research on a method for obtaining a peptide mixture of consistent quality with a low Phe content in a state that is always stable in light of the aforementioned conventional techniques, the inventors perfected the present invention upon discovering that a peptide mixture with a low Phe content is readily obtained by measuring briefly and over time the amount of Phe that is freed in the hydrolysate during the hydrolysis of the proteins using proteases, by calculating the proportion with the total amount of Phe contained in the starting material protein, by immediately stopping the hydrolysis when the value falls within a specific predetermined range, and by removing the freed Phe.
An object of the present invention is to provide a palatable whey protein hydrolysate which as exceptional gastrointestinal absorption properties and amino acid balance, which is effective in preventing and treating food allergies and has antioxidant action, which has a low ammonia content, and which can be used in a wide variety of applications, as well as to a method for producing the same.
Another object of the present invention is to provide a novel method making it possible to readily obtain a peptide mixture that has a consistent free amino acid content and composition, and that is of good quality.
Still another object of the present invention is to over a novel method that makes it possible to readily obtain a peptide mixture that is of good quality, that has a low phenylalanine content, and that can be ingested on a daily basis by patients suffering from PKU in particular.
The first of the inventions resolving the aforementioned drawbacks is a palatable whey protein hydrolysate, characterized in that the hydrolysate of whey protein having a purity of at least 70% (by weight) has the following physicochemical properties a) through h):
a) less than 1% (by weight) of the total hydrolysate consists of fractions having a molecular weight of between 5,000 and 10,000 daltons;
b) the residual antigenic activity is no more than 10xe2x88x925, as determined by the ELISA inhibition test using anti-whey protein sera;
c) the amount of free amino acids is 10 to 15% (by weight) with respect to the total amount of amino acids in the hydrolysate;
d) the amount of free lysine is 12 to 20% (by weight) with respect to the total amount of lysine contained in the whey protein;
e) the ammonia content is no more than 0.2% (by weight);
f) the transmittance of a 10% (by weight) solution is at least 98%, as determined at 540 nm using a 1-cm cell;
g) no precipitation results when a 5% (by weight) solution with pH 4 to 7 is heated for 10 minutes at 120xc2x0 C.; and
h) it has antioxidant activity.
The second of the inventions resolving the aforementioned drawbacks is a method for producing a palatable whey protein hydrolysate, characterized in that whey protein with a purity of at least 70% (by weight) is dissolved in water to a concentration of no more than 15% (by weight); the pH of the aqueous solution is adjusted to between 7.5 and 10; two types of proteases, one a Bacillus subtilis-derived endopeptidase and the other a lactic acid bacteria-derived exopeptidase, are added to the aqueous solution to initiate hydrolysis; the amount of free lysine in the hydrolysate is measured over time, and the hydrolysis is stopped when the amount of free lysine is between 12 and 20% (by weight) with respect to the total amount of lysine contained in the starting material whey protein; and fractions with a molecular weight of 10,000 daltons or more are completely removed by ultrafiltration.
The third of the inventions resolving the aforementioned drawbacks is a method for producing a peptide mixture of consistent quality, wherein said method for producing a peptide mixture is characterized in that one or more proteases is or are added to an aqueous solution of starting material proteins consisting of one or more proteins or to an aqueous solution of slightly pre-hydrolyzed starting material proteins to start the hydrolysis of the starting material protein or of the slightly pre-hydrolyzed starting material protein, the amount of a specific amino acid freed in the hydrolysate as a result of the hydrolysis is measured briefly and over time, the amount of the specific free amino acid is calculated with respect to the total amount of the specific amino acid contained in the starting material protein or in the slightly pre-hydrolyzed starting material protein, and the hydrolysis is immediately terminated when the calculated value falls within a specific predetermined range. In a preferred embodiment, the specific amino acid is lysine, phenylalanine, leucine, or arginine.
The fourth of the inventions resolving the aforementioned drawbacks is a method for producing a peptide mixture of consistent quality with a low phenylalanine content, wherein said method for producing a peptide mixture with a low phenylalanine content is characterized in that one or more proteases is or are added to an aqueous solution of starting material proteins consisting of one or more proteins or to an aqueous solution of slightly pre-hydrolyzed starting material proteins to start the hydrolysis of the starting material protein or of the slightly pre-hydrolyzed starting material protein, the amount of phenylalanine freed in the hydrolysate as a result of the hydrolysis is measured briefly and over time, the amount of the free phenylalanine is calculated with respect to the total amount of the phenylalanine contained in the starting material protein or in the slightly pre-hydrolyzed starting material protein, the hydrolysis is immediately terminated when the calculated value falls within a specific predetermined range, and the free phenylalanine in the hydrolysate is removed. In a preferred embodiment, the amount of the free phenylalanine is measured using an enzyme membrane sensor.
The present invention is described in detail below, but the description will begin with the second of the present inventions for the sake of simplicity.
The whey protein used as the starting material in the method pertaining to the present invention can be a commercially available product or the like having a purity of at least 70%. Commercially available products with a higher degree of purity, known as whey protein concentratio s (WPC) and whey protein isolates (WPI), are preferred. The whey protein is dissolved in water to a concentration of no more than 15%, and preferably to between 8 and 12%, and the pH is adjusted to between 7.5 and 10, and preferably to between 8 and 9, with an alkaline aqueous solution.
Two proteases, one being a Bacillus subtilis-derived endopeptidase and he other being a lactic acid bacteria-derived exopeptidase, are then added to the aforementioned whey protein solution. Other endopeptidases such as trypsin and papain can also be added in extremely low amounts. The addition of exopeptidass other than lactic acid bacteria-derived types should be avoided, however, because of deterioration in the taste.
Commercially available Bacillus subtilis-derived endopeptidases can be used, and are added in a proportion of 1,000 to 7,500 PUN units (the units are described below), and preferably 2,000 to 3,000 PUN units, per gram whey protein.
One PUN unit is the enzyme activity resulting in the colorimetric reaction of an allylamino acid corresponding to 1 xcexcg of tyrosine with Folin reagent after 1 minute at 30xc2x0 C. when a Bacillus subtilis-derived endopeptidase is allowed to act on casein (Hammerstein: by Merck).
A lactic acid bacteria-derived exopeptidase can be manufactured as follows by the method noted in the section entitled xe2x80x9c(3) Enzymes Usedxe2x80x9d in Japanese Patent Publication 54-36235, column 6, line 4, for example.
A lactic acid bacteria (including Bifidobacterium) is cultured by a known method (such as the method noted in Japanese Patent Publication 48-43878), the resulting broth is centrifuged to recover the lactic acid bacteria cells, the cells are suspended in sterilized water, and the lactic acid bacteria cells are recovered by centrigugation. This is repeated twice, and the cells are washed, suspended at a concentration of 20% in sterilized water, ruptured with a cell rupturing device (for example, the KDL model dyne-o-mill by Willy Bachnfen Engineering Works), and lyophilized to obtain a lactic acid bacteria-derived exopeptidase powder.
The enzyme is added in a proportion of 20 to 200 active units (the units are described below), and preferably 60 to 90 active units.
An active unit is determined by the following method. Powder containing exopeptidase is dispersed or dissolved in a proportion of 0.2 g/100 mL in 0.1 mol phosphate buffer (pH 7.0) to prepare an enzyme solution. Leucyl para-nitroanilide (by Kokusan Kaguku; henceforth Leu-pNA) is meanwhile dissolved in 0.1 mol phosphate buffer (pH 7.0) to prepare a 2 mM substrate solution. 1 mL substrate solution is added to 1 mL enzyme solution, a reaction is brought about for 5 minutes at 37xc2x0 C., and the reaction is then stopped with the addition of 2 mL of 30% acetic acid solution. The reaction solution is filtered with a membrane filter, and the filtrate absorbance is determined at a wavelength of 410 nm. One active unit of exopeptidase is defined as the amount of enzyme needed to degrade 1 (mol of Leu-pNA in 1 minute, as determined by the following formula.
Active unit (per gram powder)=20xc3x97(A/B)
A and B in the above formula indicate the absorbance of the sample and the absorbance of 0.25 mM para-nitroaniline, respectively, at a wavelength of 410 nm.
The solution to which the enzyme has been added is maintained at 30 to 60xc2x0 C., and preferably 45 to 55xc2x0 C., to initiate the hydrolysis of the whey protein. When the pH falls as the hydrolysis progresses, the pH should be kept at 6 or more, and preferably between 6 and 7.
After the hydrolysis has been initiated, the amount of free lysine in the hydrolysate is measured over time using a device capable of measuring the amount of free lysine in the hydrolysate over time, such as a Biotech Analyzer (by Asahi Kasei Kogyo), and when the proportion of free lysine is within 12 to 20%, and preferably 14 to 17%, with respect to the total amount of lysine contained in the starting material whey protein, the reaction liquid is immediately heated (for example, 15 minutes at 85xc2x0 C.) to inactivate the enzyme and stop the hydrolysis.
The resulting reaction solution is adjusted to a pH of between 5.5 and 7 with an acid such as citric acid and is subjected to ultrafiltration using a well-known device (such as the Ultrafiltration Module by Asahi Kasei Kogyo), so as to completely remove fractions with a molecular weight of 10,000 daltons or more and obtain the targeted, palatable whey protein hydrolysate. The liquid containing the whey protein hydrolysate can be concentratio d by a well-known method to produce a concentrated liquid, and the concentratio d liquid can also be dried by a well-known method to make a powder.
The whey protein hydrolysate obtained in the manner described above has the following physicochemical properties, as will be elucidated in the practical examples below.
a) As indicated in FIG. 1, less than 1% (by weight) of the total hydrolysate consists of fractions having a molecular weight of between 5,000 and 10,000 daltons. It contains no fractions with a molecular weight of 10,000 daltons or more. At least 70% of the fractions have a molecular weight of less than 1,000 daltons. It has peaks at a molecular weight of 500 daltons and a molecular weight of 1,000 daltons. The number average molecular weight is 300 to 400 daltons, and the weight average molecular weight is 600 to 800 daltons.
FIG. 1 indicates the molecular weight distribution of the whey protein hydrolysate of the present invention obtained in Example 1. The vertical and horizontal axes indicate the distribution ratio and the molecular weight, respectively.
b) As shown in FIG. 2, the residual antigenic activity is no more than 10xe2x88x925, and preferably no more than 10xe2x88x926, as determined by the ELISA inhibition test using anti-whey protein sera.
FIG. 2 indicates the residual antigenic activity of the whey protein hydrolysate of the present invention obtained in Example 1. The vertical and horizontal axes indicate the inhibiting ratio and final sample concentration, respectively. In the figure, plus signs and boxes indicate the whey protein hydrolysate of the present invention and whey protein, respectively.
c) The amount of free amino acids is 10 to 15% (by weight), and preferably 11 to 13% (by weight), with respect to the total amount of amino acids in the hydrolysate.
d) The amount of free lysine is 12 to 20% (by weight), and preferably 14 to 17% (by weight), with respect to the total amount of lysine contained in the whey protein.
e) The ammonia content is no more than 0.2% (by weight), and preferably no more than 0.1% (by weight).
f) The transmittance of a 10% (by weight) solution is at least 98%, as determined at 540 nm using a 1-cm cell.
g) No precipitation results when a 5% (by weight) solution with pH 4 to 7 is heated for 10 minutes at 120xc2x0 C.
h) As shown in FIG. 3, it has antioxidant activity equal to or greater than that of xcex1-tocopherol, a well-known antioxidant. FIG. 3 indicates the antioxidant activity of the whey protein hydrolysate of the present invention obtained in Example 1. The vertical and horizontal axes indicate the residual antioxidant capacity and the time, respectively. In the figure, the diamonds, plus signs, and boxes indicate the whey protein hydrolyate of the present invention, xcex1-tocopherol, and a control (sample or preparation added), respectively.
The third of the inventions is described below.
The starting proteins used as starting material in the method pertaining to the present invention are not particularly limited and include animal proteins (such as those derived from animal milk, eggs, fish, meat, and the like), vegetable proteins (such as those derived from grains, seaweed, rice, and the like), and any mixture of these. Peptide mixtures of substantial molecular weight which are hydrolysates of slightly pre-hydrolyzed proteins and which can be further hydrolyzed proteases can be used as starting material.
The starting material aqueous solution is prepared by dissolving the starting protein or slightly pre-hydrolyzed starting protein in water to a concentration of around 10%, calculated in terms of protein, and by adjusting the solution pH with an alkali solution or acid solution to around the optimal pH of the protease being used.
Animal-derived (such as pancreatin, pepsin, trypsin, and the like), vegetable-derived (such as papain, bromelain, and the like), microbe-derived (such as mold, actinomyces, bacteria, lactic acid bacteria, or the like) proteases, or any combination of these, may be selected as desired according to the intended use and added in the prescribed amounts. In a preferred embodiment, for example, the endopeptidase can be added in an amount of 2000 to 5000 PUN units per gram starting protein, and the exopeptidase can be added in an amount of 20 to 100 active units per gram starting protein.
The starting material aqueous solution to which the prescribed amounts of enzymes have been added is usually maintained for a prescribed time at the optimal temperature of the enzymes to bring about the zymolysis of the protein. When microbial growth is a concern during the hydrolysis, the solution can be maintained as needed for a prescribed time at a temperature higher or lower than the optimal temperature of the enzymes to bring about the zymolysis of the protein.
The hydrolysis is initiated, and the amount of a specific amino acid in the hydrolysate is measured briefly and over time. Specifically, this can be done using, for example, an HPLC, a Biotech Analyzer (by Asahi Kasei Kogyo), a perfusion chromatograph (BioCAD by Perceptive Biosystems), and the like. Because the amount of amino acid that is freed varies, depending on the type of starting protein ad enzyme used, amino acids that are freed in large amounts should be selected as the specific amino acid. In a particularly preferable embodiment, the amount of specific amino acid freed in the hydrolysate can be measured on-line. Examples of specific amino acids that are particularly preferred include lysine, phenylalanine, leucine, and arginine. In this way, the amount of the specific amino acid freed in the hydrolysate is measured briefly and over time, and when the proportion of the specific free amino acid falls within a specific predetermined range with respect to the total amount of the specific amino acid contained in the starting material protein, the enzymes in the reaction solution are immediately inactivated or removed to stop the hydrolysis. The aforementioned specific range varies, depending on the target peptide mixture, the starting material that is used, the enzymes that are used, and the like, but when, for example, the whey protein is hydrolyzed as the amount of free lysine is measured briefly and over time, a range of 5 to 40% can be given as an example of the range for the amount of free lysine.
The method for inactivating or removing the enzymes in the reaction liquid to stop the hydrolysis is not particularly limited in the present invention, and a desired method may be used, although there are cases where there are time lags until the hydrolysis is stopped (for example, it sometimes takes 30 to 60 minutes to heat a certain amount of hydrolysate until the enzyme is inactivated). In such cases, because of the risk of continuing hydrolysis, pre-tests should be conducted in advance, the ratio at which the hydrolysis progresses (for example, the velocity at which the specific free amino acid is produced) should be measured based on prescribed conditions, and the time needed to inactivate or remove the enzyme should be taken into account to determine and establish the aforementioned specific range.
The hydrolysate containing the completely hydrolyzed peptide mixture can be concentrated by a common method to make a concentrated liquid, and the concentratio d liquid can be dried by a common method to make a powder. The liquid containing the peptide mixture can also be purified by a common method such as ultrafiltration and gel filtration and concentratio d by a common method to make a concentratio d liquid, and the concentrated liquid can be dried by a common method to make a powder.
The configuration of the reaction vessel in which the hydrolysis is carried out (such as tanks, tubes, columns, or the like), the mode of hydrolysis (such as batch, continuous, or consecutive modes), the method for inactivating, separating, or removing the enzymes, the method for purifying the peptides, and the like are not particularly limited, and common methods and devices can be used.
A peptide mixture of consistent quality, in which the free amino acid content and composition are always constant, can be manufactured in the manner described above.
The fourth of the inventions is described below.
The starting protein used as starting material in the present invention is not particularly limited and may be an animal protein derived from animal milk, eggs, fish, meat or the like, vegetable proteins derived from soybeans, wheat, or the like, or any mixture of these. Peptides mixtures of substantial molecular weight which are hydrolysates of slightly pre-hydrolyzed proteins and which can be further hydrolyzed by proteases can be used as starting material.
The starting protein or slightly pre-hydrolyzed starting protein is dissolved in water to a concentration of around 10%, calculated in terms of protein, and a 5 to 30 minute heat treatment at a temperature ranging between 65 and 90xc2x0 C. may be undertaken to ensure efficient sterilization and zymolysis of the proteins. The solution pH is then adjusted with an alkali solution or acid solution to around the optimal pH of the protease being used so as to prepare the starting material aqueous solution.
The protease is then added to the aforementioned starting material aqueous solution. The protease may be added all at once or consecutively by being added a little at a time. Examples of proteases which are used include animal-derived (such as pancreatin, pepsin, trypsin, chymotrypsin, and the like), vegetable-derived (such as papain, bromelin, and the like), and microbe-derived (such as yeast, mold, bacteria, actinomyces, lactic acid bacteria, or the like) endoproteases and exoproteases. Those having affinity for aromatic amino acids such as Phe are preferred as endoproteases. Examples include pepsin, chymotrypsin, and the like. Those showing peptidase activity for peptides that have an aromatic amino acid such as Phe at the terminal are preferred as exoproteases. Desirable examples which can be used include exopeptidases derived from Aspergillus, Actinomyces, yeast, and the like.
To ensure that enough Phe is freed, the aforementioned endoprotease and exoprotease should be used in combination. The hydrolysis can be brought about by adding the enzymes all at once to the starting protein solution, or in steps by adding the endoprotease, and then adding the exoprotease after a certain time has elapsed. In a preferred embodiment, for example, the enzymes are added in amounts such that the endopeptidase is added in an amount of 5000 to 10000 PUN units per gram starting protein, and the exopeptidase is added in an amount of 10 to 50 active units per gram starting protein.
The starting material aqueous solution to which the prescribed amounts of enzymes have been added is usually maintained for a prescribed time at the optimal temperature of the enzymes to bring about the zymolysis of the protein. When microbial growth is a concern during the hydrolysis, the solution can be maintained as needed for a prescribed time at a temperature higher or lower than the optimal temperature of the enzymes to bring about the zymolysis of the protein. However, the temperature should usually range from 40 to 60xc2x0 C. because the hydrolysis efficiency decreases in a temperature range that differs significantly from the optimal temperature of the enzymes.
After the hydrolysis is initiated, the amount of a Phe freed in the hydrolysate is measured briefly and over time. This can be done using, for example, an HPLC, an enzyme membrane sensor (such as a Biotech Analyzer (by Asahi Kasei Kogyo)), and the like. In a particularly preferable embodiment, a method of on-line measurement can be used. In this way, the amount of Phe freed in the hydrolysate is measured briefly and over time, and when the proportion of the free Phe falls within a specific predetermined range with respect to the total amount of the Phe contained in the starting material protein or slightly pre-hydrolyzed protein, the enzymes in the hydrolysate are immediately inactivated or removed to stop the hydrolysis. Because the Phe contained in the starting protein or slightly pre-hydrolyzed protein must be freed and removed at high levels to provide a peptide mixture for patients suffering from PKU, the aforementioned specific range can be 85 to 95%, and preferably 88 to 92%.
The enzymes can be inactivated or removed from the hydrolysate using common heat treatments, ultrafiltration, or the like. Temperature and time conditions which allow the enzymes to be sufficiently inactivated in the heat treatment can be established as desired in view of the thermal stability of the enzymes that are used. When a certain time is needed to inactivate or remove the enzymes by heat treatment or ultrafiltration, during which there is a chance that the hydrolysis will continue, the ratio at which the hydrolysis progresses (for example, the velocity at which the Phe is freed) should be tested in advance based on certain conditions, and the aforementioned specific range in which the hydrolysis is to be stopped should be determined and established on the basis of the results.
After the enzymes in the hydrolysate have been inactivated or removed, the hydrolysate is cooled by a common method, and the precipitate is removed from the hydrolysate by a method such as celite filtration, precision filtration, ultrafiltration, or centrifugation. The aromatic amino acids such as Phe can be removed from the resulting hydrolysate by a desired method such as gel filtration, an adsorptive resin method, an activated carbon adsorption method, or the like, either alone or in combination. A gel filtration agent with a molecular weight cut-off of 10,000 daltons or less, and preferably 2,500 daltons or less, is used, and the use of a gel carrier having aromatic amino acid-adsorbing hydrophobic side chains, such as carboxyl groups, butyl groups, phenyl groups, and hydrophobic sites, is particularly preferred. Examples of such gel filtration agents include Sephadex G-10 (by Pharmacia) and Cellulofine GCL-25 (by Seikagaku Kogyo), and examples of activated carbon include Sirasagi (by Takeda Yakuhinn Kogyo).
A column is packed with the gel filtration agent or activated carbon, and the hydrolysate is allowed to flow through the column. Water can be used as the eluate, or a 2 to 15% ethanol aqueous solution can be used to enhance the aromatic amino acid adsorption. The aromatic amino acids can be removed by a batch method in which the gel filtrate agent or activated carbon is placed in the hydrolysate and allowed to stand for a prescribed time so as to adsorb the aromatic amino acids.
The solution of the peptide mixture with a low phenylalanine content obtained by the aforementioned method can then be concentratio d by a common method to make a concentratio d liquid, and the concentrated liquid can be dried by a common method to make a powder. The solution, concentratio d liquid, or powder of the peptide mixture with a low phenylalanine content thus obtained can be used as starting material for food products designed for patients suffering from PKU in the same way as ordinary food product starting materials so as to manufacture a variety of food products for patients suffering from PKU.